Mutation Screening Using Automated

The need continues to grow for mutation identification in genetic disease in both research and clinical settings. We have developed a rapid nonradioactive bidirectional dideoxy fingerprint mutation screening procedure that is performed using an automated DNA analyzer. This technique features standardized primers and easily interpreted results from separate, but simultaneously collected, images for coding and noncoding strands. Another advantage is simplified mutation verification by sequencing using the same amplified DNA templates and also application to large multi-exon genes. We demonstrate the efficiency and reproducibility of the method in which we screen a DNA fragment encompassing exon 5 of the PTCH gene (in which mutations cause Gorlin Syndrome) in a panel of 22 patients. INTRODUCTION As researchers identify gene defects in human diseases, there are increasing expectations from patients that mutation detection for diagnosis and genetic counseling will be made available to the medical community. Mutation testing represents a real opportunity for families with genetic disease to achieve better control of their own destinies. In disease genes in which the defects may be due to one or only a few specific mutations, sequence-specific tests have been developed using, for example, allelespecific amplification, restriction enzyme digestion or other advanced techniques such as ligase chain reaction and biochips. For other diseases that arise from a mutation that could occur anywhere within a gene, screening of the entire gene is required. Sequencing, while virtually 100% effective at finding mutations, remains technically challenging, resource and labor-intensive and is generally the least cost-effective screening technique to perform. Alternative mutation screening methods that are efficient, rapid, high-throughput and automated, are better suited to the needs of clinical laboratories where time is critical and high sensitivity is crucial. Single-stranded conformational polymorphism analysis (SSCP) is one of the most common methods used for mutation screening (5). It is based on detecting changes in the electrophoretic mobility of partially re-annealed DNA fragments caused by sequence variation. Despite improvements, SSCP remains only 60%–90% efficient at uncovering mutations (8) and is achievable only for short DNA fragments (~ 200 bp). The method requires that electrophoresis conditions be optimized for each unique fragment and that they need to be performed at a low temperature (such as 4°C). Heteroduplex analysis is another screening approach that detects mobility changes between re-annealed complementary and mismatched doublestranded PCR fragments (10). It is less efficient than SSCP and has a similar range of problems with its implementation. The combination of SSCP and heteroduplex analysis has improved mutation detection but still did not reach 100% efficiency (9). The most effective screening technique available may be dideoxy fingerprinting (ddF; see Reference 8), which combines dideoxynucleotide sequencing with SSCP analysis. In ddF, fragments generated by a single dideoxy sequencing reaction (i.e., ddGTP) with 32P-labeled primers are resolved on an SSCP-type gel. Changes in the banding pattern from sequence variations are readily detected by an inspection of control and test samples run side by side. This method has been found to detect 100% of mutations when both strands of the target sequence are evaluated (8). An important improvement was the introduction of a bidirectional dideoxy fingerprint (Bi-ddF) protocol, in which both strands are examined simultaneResearch Report 134 BioTechniques Vol. 28, No. 1 (2000) Mutation Screening Using Automated Bidirectional Dideoxy Fingerprinting BioTechniques 28:134-138 (January 1999)